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Pef1 v5 his vector

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The PEF1/V5-His vector is a plasmid used for the expression of recombinant proteins in mammalian cells. It contains a CMV promoter, a multiple cloning site for inserting the gene of interest, and a V5-His tag sequence for detection and purification of the expressed protein.

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Lab products found in correlation

Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (3 mentions) Mmessage mmachine, by Thermo Fisher Scientific (3 mentions) Er tracker, by Thermo Fisher Scientific (1 mentions) Apyrase, by Merck Group (1 mentions) α tubulin, by Merck Group (1 mentions) Ly294002, by Thermo Fisher Scientific (1 mentions) P nfatc1, by Santa Cruz Biotechnology (1 mentions) Iq sybr green supermix, by Bio-Rad (1 mentions) Nfatc1, by BD (1 mentions)

5 protocols using pef1 v5 his vector

1

In Vitro Transcription of Mutant TET2 and Other Zebrafish Genes

Cited 1 time
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The human TET2 vector used for mRNA production has been previously described (Li et al., 2015 (link)). Briefly, the human TET2 ORF corresponding to GenBank: NM_001127208 was amplified from cDNA made from SH-SY5Y neuroblastoma cells. Following sub-cloning, the TET2 ORF was introduced into the pEF1/V5-His vector (Invitrogen) to allow for in vitro transcription. Mutant TET2 (H1382Y, D1384A) was generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). The inhbaa, sox9b and has2 ORF were PCR amplified from a 2 dpf zebrafish embryo cDNA library and cloned into the pCS2+ vector. Sequences of all clones were confirmed by conventional DNA sequencing. PCR primers used are listed in Table S1. Capped RNA was synthesized using mMESSAGE mMACHINE (Invitrogen) with Sp6 polymerase. For each experimental condition, mRNA was injected into at least 100 embryos derived from tet2mk17/mk17, tet3mk18/+ intercrosses.
Lan Y., Pan H., Li C., Banks K.M., Sam J., Ding B., Elemento O., Goll M.G, & Evans T. (2019). TETs Regulate Proepicardial Cell Migration through Extracellular Matrix Organization during Zebrafish Cardiogenesis. Cell reports, 26(3), 720-732.e4.
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2

In Vitro Transcription of Mutant TET2 and Other Zebrafish Genes

Cited 1 time
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The human TET2 vector used for mRNA production has been previously described (Li et al., 2015 (link)). Briefly, the human TET2 ORF corresponding to GenBank: NM_001127208 was amplified from cDNA made from SH-SY5Y neuroblastoma cells. Following sub-cloning, the TET2 ORF was introduced into the pEF1/V5-His vector (Invitrogen) to allow for in vitro transcription. Mutant TET2 (H1382Y, D1384A) was generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). The inhbaa, sox9b and has2 ORF were PCR amplified from a 2 dpf zebrafish embryo cDNA library and cloned into the pCS2+ vector. Sequences of all clones were confirmed by conventional DNA sequencing. PCR primers used are listed in Table S1. Capped RNA was synthesized using mMESSAGE mMACHINE (Invitrogen) with Sp6 polymerase. For each experimental condition, mRNA was injected into at least 100 embryos derived from tet2mk17/mk17, tet3mk18/+ intercrosses.
Lan Y., Pan H., Li C., Banks K.M., Sam J., Ding B., Elemento O., Goll M.G, & Evans T. (2019). TETs Regulate Proepicardial Cell Migration through Extracellular Matrix Organization during Zebrafish Cardiogenesis. Cell reports, 26(3), 720-732.e4.
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3

Antibody Characterization of Panx3 Protein

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A rabbit polyclonal P-Panx3 antibody was raised against a synthetic peptide (amino acid residues, SCFpSPSNFSC) from the first extracellular loop of the mouse Panx3 protein and the native peptide was used as a control sequence (SCFSPSNFSC). Rabbit anti-Panx3 antibody was used as previously described5 (link),16 (link). The Panx3 expression vector (pEF1/Panx3) and the control vector (pEF1) have been described previously16 (link). In brief, the pEF1/Panx3 vector was constructed by cloning the coding sequence of mouse Panx3 cDNA into the pEF1/V5-His vector (Invitrogen). The antibodies were obtained for Ocn from Biomedical Technology; CaMKII and P-CaMKII from Cell Signaling Technology; P-NFATc1 from Santa Cruz Biotechnology, Inc.; V5 from Invitrogen; and NFATc1 from BD. HA was from COVANCE; α-tubulin from Sigma-Aldrich, CIP from New England Biolabs; Apyrase from Sigma-Aldrich; LY294002 from Invitrogen; BMP2 from Humanzyme; and iQ SYBR Green Supermix from Bio-Rad Laboratories. ER-tracker was obtained from Invitrogen. HRP-conjugated goat anti–mouse and goat anti–rabbit IgG were obtained from United States Biological. The Akt-CA was obtained from Addgene. The inhibitory Panx3 peptide was described previously16 (link).
Ishikawa M., Williams G., Forcinito P., Ishikawa M., Petrie R.J., Saito K., Fukumoto S, & Yamada Y. (2019). Pannexin 3 ER Ca2+ channel gating is regulated by phosphorylation at the Serine 68 residue in osteoblast differentiation. Scientific Reports, 9, 18759.
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4

Transcription Factor Overexpression in Zebrafish

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The human TET3 vector used for mRNA production has been previously described (Ko et al., 2013 (link)). The human TET2 ORF corresponding to Genebank: NM_001127208 was was amplified from cDNA made from SH-SY5Y neuroblastoma cells. Following sub-cloning, the TET2 ORF was introduced into the pEF1/V5-His vector (Invitrogen) to allow for in vitro transcription. Mutant TET2 (H1382Y, D1384A) was generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). pExpress-1-gata2b was purchased from Transomic Technologies. For scl-β, RT-PCR amplified scl-β cDNA with sequence corresponding to Genbank: EF488003 was cloned into pCS2+. Sequences of all clones were confirmed by conventional DNA sequencing. In all cases, capped RNA was synthesized using mMessage mMachine (Ambion) with Sp6 or T7 polymerase as appropriate to the vector. For each experimental condition, mRNA was injected into at least fifty embryos derived from tet2mk17/mk17, tet3mk18/+ intercrosses.
Li C., Lan Y., Schwartz-Orbach L., Korol E., Tahiliani M., Evans T, & Goll M.G. (2015). Overlapping requirements for Tet2 and Tet3 in normal development and hematopoietic stem cell emergence. Cell reports, 12(7), 1133-1143.
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5

Generating Jpx and JPX Constructs

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The Tg(EF1α:JpxE1-E3) construct was generated by PCR-cloning the Jpx transcript out of the cDNA library prepared from the total RNA of differentiated mES cells. The Jpx E1-E3 isoform was cloned into pEF1/V5-His vector (Invitrogen Cat# V92020), which contains an EF-1α promoter for mammalian expression and a T7 promoter for in vitro transcription. In parallel, the Tg(EF1α:JPXE1-E3) construct was generated by PCR-cloning the JPX transcript out of the cDNA prepared from the total RNA of human SKOV3iP1 ovarian cancer cells. The JPX E1-E3 isoform was cloned into the pEF1/V5-His mammalian expression vector the same way as Jpx E1-E3.
Karner H., Webb C.H., Carmona S., Liu Y., Lin B., Erhard M., Chan D., Baldi P., Spitale R.C, & Sun S. (2020). Functional Conservation of LncRNA JPX Despite Sequence and Structural Divergence. Journal of molecular biology, 432(2).
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